Genes and networks associated with circadian rhythms

Arabidopsis thaliana, a model plant in research, possesses the most extensively characterized circadian network (Fig. 1A and B). Through forward and reverse molecular genetics, it has been elucidated that the circadian rhythm in plants is tightly controlled by multiple interlocked transcriptional–translational feedback loops (TTFL) (Hernando et al. 2017; McClung 2019; Patnaik et al. 2022). A key TTFL is composed of the genes Circadian Clock Associated 1 (CCA1), Late Elongated Hypocotyl (LHY), and Timing of CAB2 Expression 1 (TOC1). CCA1 and LHY are partially redundant MYB-related Transcription Factors (TFs) that belong to the Reveille (RVE) gene family, where both are expressed at dawn; i.e. during the onset of the light phase of day–night cycle (Lu et al. 2009; Schaffer et al. 1998; Wang et al. 1997). In contrast, TOC1 belongs to the pseudo-response regulator (PRR) gene family expressed in the evening (Gendron et al. 2012). Both CCA1 and LHY proteins can hetero- and homo-dimerize (Lu et al. 2009; Yakir et al. 2009), and negatively regulate gene expression by interacting with the “Evening Elements” (EE, AAAATATCT) and “CCA1-binding sites” (CBS) on the promoter region of its own genes, of TOC1 and other circadian clock genes (Carre and Kay 1995; Harmer et al. 2000; Nagel et al. 2015; Wang et al. 1997). TOC1 also acts as a transcription repressor of itself and other circadian regulatory components (Gendron et al. 2012; Huang et al. 2012; Pokhilko et al. 2012). TOC1 represses the expression of CCA1 by interacting with a Teosinte Branched1-Cycloidea-PCE (TCP) protein, the CCA1 Hiking Expedition (CHE) TF (Pruneda-Paz et al. 2009). Thus, CCA1/LHY and TOC1 represent the core negative TTFL in the A. thaliana circadian clock, where the morning-expressed CCA1/LHY represses the transcription of the evening-expressed TOC1, while TOC1 inhibits the expression of CCA1/LHY during the evening.

Fig. 1
figure 1

Circadian oscillator components. A Extended model representation of the components of the circadian oscillator. B Simplified model representation of the components of the circadian oscillator. Solid black lines ending with dashes represent the transcriptional repression. C Graphic representation between the hormonal response and the circadian oscillator. The relative timing of action to every component is illustrated in the upper part of the extended model, from left to right during a day–night cycle. Transcriptional repressors are filled in dark blue colour, while transcriptional activators are filled in green colour. Solid lines represent direct regulatory effects while dashed lines represent indirect (or possibly direct but not experimentally determined) effects. Black lines ending with dashes represent the transcriptional repression; green lines ending with arrows represent the transcriptional activation. The circadian repressor components are Circadian Clock Associated 1 (CCA1); Late Elongated Hypocotyl (LHY); Timing of CAB2 Expression 1 (TOC1); Pseudo-Response Regulator 9 (PRR9), PRR7 and PRR5; evening complex (EC) components formed by LUX Arrhythmo (LUX) or Brother of LUX Arrhythmo (BOA), Early Flowering 3 (ELE3), and ELE4; Cold-Regulated 27 (COR27) and COR28; CCA1 Hiking Expedition (CHE). The circadian activation components are Reveille (RVEs); Night Light-Inducible and Clock-Regulated 1 (LNK1), and LNK2; Light-Regulated WD1 (LWD1) and LWD2; Teosinte Branched1-Cycloidea-PCE 20 (TCP20), and TCP22. Proteins involved in brassinosteroids (BRs) circadian regulations are bri1-EMS-Suppressor 1 (BES1) and Topless (TPL). Proteins involved in the Abscisic acid (ABA) circadian regulation are biosynthesis 9-cis-epoxycarotenoid dioxygenase enzymes 3 (NCED3) and MYB96. In the Jasmonic acid (JA) circadian regulation, a basic helix-loop-helix-leucine zipper transcription factor (MYC2) is a key component. Regarding auxin regulation, RVE1 (a member of the RVE transcription factor family but not involved in the core circadian regulation) as well as the auxin biosynthetic gene YUCCA8 (YUC8) are involved

Full size image

In addition to TOC1, other PRR proteins are involved in circadian rhythm oscillations. PRR9, PRR7, PRR5 and TOC1 are consecutively expressed with moderate differences in their peak expression (Gendron et al. 2012; Liu et al. 2013; Matsushika et al. 2000; Michael et al. 2003; Nakamichi et al. 2010, 2012). PRR9, PRR7 and PRR5 also act as transcription repressors, and together with TOC1, repress the previously expressed PRRs and collectively restrict the transcription of CCA1 and LHY to a period during the dawn (Liu et al. 2013; Nakamichi et al. 2010, 2012). In turn, CCA1/LHY transcriptionally represses PRR9 and PRR7 during the dawn (Adams et al. 2015; Fogelmark and Troein 2014; Greenwood et al. 2022; Joanito et al. 2018; Kamioka et al. 2016; Shalit-Kaneh et al. 2018). Another key component of the clock that is transcriptionally repressed by CCA1/LHY and TOC1 is the evening complex (EC) (Gendron et al. 2012; Huang and Nusinow 2016). The EC is composed of the two plant-specific proteins Early Flowering 3 (ELF3) and Early Flowering 4 (ELF4) and the Lux Arrhythmo (LUX), a MYB-like TF that belongs to the GARP protein family or its close homolog Brother of Lux Arrhythmo (BOA) (Dai et al. 2011; Herrero et al. 2012; Nusinow et al. 2011). The EC complex is expressed after dusk and acts as a transcriptional repressor that binds to the promoter regions of PRR7, PRR9 and LUX (Chow et al. 2012; Dixon et al. 2011; Helfer et al. 2011). LUX (or BOA) is the EC component with DNA-binding capability, since ELF3 and ELF4 do not bind to DNA (Huang and Nusinow 2016). Other corepressors involved in the TTFL are COLD-Regulated 27 (COR27) and COR28, which are repressed by CAA1 and repress PRR5 and TOC1 (Li et al. 2016; Wang et al. 2013; Rawat et al. 2011). These RVEs interact with the coactivators Night Light-Inducible and Clock-Regulated 1 (LNK1) and LNK2 to form a complex and activate the transcription of TOC1, PRR5 and ELF4 (Rugnone et al. 2013; ** system, circadian clock genes generate approximately 24-h rhythms in physiology and behavior. The detection of clock genes can be based on two complementary approaches. The forward-genetic approach is based on arhythmic mutants that produce rhythms deviating from 24-h cycles. Although this approach has been instrumental for revealing the detailed molecular mechanisms of circadian rhythms in model systems and various outcrossing species (King and Takahashi 1996; Miyoshi et al. 2019), it has some significant drawbacks. First, its translational relevance for a broad spectrum of species and biological processes is challenging since only a limited number of mutations can be identified. Also, many clock genes are not evolutionarily conserved; e.g., some clock genes are species-specific (Calixto et al. 2016). This suggests that ideally, detection of clock genes should be based on individual species. To overcome these drawbacks, a forward-genetic approach has been developed which takes advantage of genome-wide map** and association genetics available for a range of species (Jones et al. 2019). Such an approach could identify a more complete set of clock genes. For example, using a map** population, a number of QTL for circadian clock parameters, such as period, phase, and amplitude, were identified in Arabidopsis thaliana (Rubin et al. 2019). A review of progress in clock QTL map** and its application to understanding the genetic architecture of plant adaptation is available (Anwer and Quint 2022).

Most clock map** attempts have been based on genotype–phenotype association analysis using markers. However, the results from these analyses remain an approximation of reality as the circadian rhythm is complex, involving interactions of many genes with environment. A computational algorithm to map a complete set of clock genes using association genetics has been developed (Sun et al. 2021). This algorithm is powerful in terms of its capacity to reconstruct omnigenic interactome networks for rhythmic traits (Sun et al. 2021). Omnigenic theory predicts that all genes that an organism may carry can be involved in controlling a complex trait developed through gene–gene interaction networks (Boyle et al. 2017). Factoring epistasis interactions (i.e., epiGWAS) could resolve some of the missing heritability for complex traits often found in GWAS or biparental populations (Slim et al. 2020). The algorithm can coalesce all genes into bidirectional, signed, and weighted networks, allowing for the characterization of a roadmap of how each and every gene affects circadian rhythms through direct and indirect pathways. It can also discern how each gene functions. First, some clock genes are significant due to their own strong intrinsic capacity, thus implying that these genes govern circadian rhythms without relying on the promotion of other regulators. Second, the significance of some genes is due to favorable regulation involving other genes. The practical use of these two gene types to improve rhythmic traits should be based on different strategies. In addition, some genes are detected to be insignificant not because their effects are inherently small but because they are inhibited by negative regulators. Thus, the effects of these insignificant genes can be released by knocking down the negative regulators. In theory, this is also an approach for retrieving missing heritability, a common phenomenon detected in current genome-wide association studies. The statistical robustness of this approach has been confirmed (Sun et al. 2021) and also its biological value has been validated by analyzing periodic gene expression data of the malaria parasite Plasmodium falciparum (Smith et al. 2020) and growth trajectory data under contrast environmental conditions in a woody plant, Populus euphrates (Wang et al. 2021).

Non-invasive, high throughput assays to measure circadian rhythms

Lack of high throughput protocols has hindered progress in investigating circadian dynamics where such assays were either manually intensive, or of low throughput, technologically expensive, or required genetic modification (Ford et al. 2016; Nagano et al. 2012; Sugiyama et al. 2001; Xu et al.

Circadian control of phytohormone biosynthesis and signaling sha** plants to external environments

Phytohormones are signaling molecules present in plants that occur in extremely low concentrations and play a major role in plant growth, development, reproduction, and fitness. Abscisic acid (ABA), ethylene (ET), jasmonates (JA), and salicylic acid (SA) are some of the stress-responsive phytohormones, while auxin (AUX), brassinosteroids (BRs), cytokinins (CK), and gibberellins (GA) are growth promoting phytohormones (Gray 2004; Verma et al. 2016). Phytohormones control specific features of the plant–circadian system, though in distinct ways. CK delays circadian phase, AUX regulates circadian amplitude and clock precision, and BR and ABA modulate circadian periodicity. This indicates that plants have multiple input/output feedbacks to adjust the clock to external signals and accurately maintain the clock system (Hanano et al. 2006). The circadian clock interconnects with an intricate network of different pathways, including phytohormones (Singh and Mas 2018).

Arabidopsis has been extensively investigated to unlock the molecular basis of connecting the circadian rhythms and phytohormone pathways in response to endogenous and exogenous cues (Fig. 1C). A complex interaction between the circadian clock and ABA pathways in Arabidopsis regulates plant responses to abiotic stress tolerance. Late Elongated Hypocotyl (LHY) transcription factor (TF) modulates ABA biosynthesis and ABA responses, which drives the rhythmic accumulation of ABA to ensure peak accumulation of phytohormones when water deficit is most severe. LHY gene represses 9-cis-epoxycarotenoid dioxygenase enzymes, a rate-limiting step of ABA biosynthesis. Plants overexpressing LHY show reduced levels of ABA under drought, whereas loss-of-function mutants exhibited altered rhythm of ABA accumulation. LHY also binds the promoter of multiple components of ABA signaling pathways and regulates responses downstream of the phytohormone. LHY promotes the expression of ABA-responsive genes involved in stress tolerance while inhibiting the negative effect of ABA on seed germination and plant growth (Adams et al. 2018).

Circadian clock gates respond to environmental signals to maximize plant fitness. Clock gating is the clock’s regulation of signaling pathways in a manner that the same stimuli given at different time points result in different responses, usually different intensities, which help plants to reduce stress responses at certain times of the day to ensure optimized growth. The MYB96 TF connects with the clock oscillator to shape the circadian gating of ABA responses by directly binding to the TOC1 promoter to regulate its expression as evidenced in Arabidopsis myb96 and toc1-3 mutants. CCA1 may also regulate MYB96-TOC1 function to alter its circadian expression. Thus, a complex circuitry of CCA1-MYB96-TOC1 regulatory interactions provides the mechanistic basis to connect circadian clock and stress signaling to optimize plant defense and fitness (Lee et al. 2016). BRs control many physiological processes via extensive cross-talks with diverse signaling networks. The BR-activated TF bri1-EMS-Suppressor 1 (BES1) together with TPL represses CCA1 and LHY expression at night by binding to their promoters. This repression by BR treatment is compromised in bes1-ko and tpl8 mutants. Consistent BRs treatment for an extended periods shorten the circadian period, where BR-induced rhythmic shortening was impaired in bes1-ko and tpl8 single mutants as well as in the double mutant, cca1-1lhy-21, providing evidence that BES1/TPL-CCA1/LHY module shapes circadian gating of BR signaling in plants (Lee et al. 2020).

Leaf senescence is associated with plant ageing or induced by external stresses to relocate nutrients from aging tissues to juvenile and reproductive organs. JA, an endogenous signal, induces leaf senescence (Sobieszczuk-Nowicka et al. 2018). The evening complex (EC), a core component of the circadian oscillator, negatively regulates leaf senescence. EC is involved in JA signaling and response as evidenced by accelerated leaf senescence in EC mutants upon JA induction. EC binds MYC2 promoter to repress its expression. MYC2 is the key activator of JA-induced leaf senescence as in EC triple (myc2 myc3 myc4) mutants. The accelerated JA-induced leaf senescence in EC mutant is abrogated by myc2 myc3 myc4 triple mutation, providing direct evidence that a core component of the circadian clock gates JA signaling to regulate leaf senescence (Zhang et al. 2018b). JA signaling regulates multiple outputs of plant defense and growth to minimize adverse effects of growth-defense trade-offs in complex environments (Li et al. 2022).

Auxin plays a central role in plant growth and development in response to environmental cues. RVE1 is a Myb-like clock regulated TF homologous to CCA1 and LHY. Its inactivation has no effect on circadian rhythmicity but instead causes a growth phenotype, indicating it as a ‘clock output’ that affects plant development. CCA1 regulates growth via bHLH TFs PIF4 and PIF5. However, RVE1 acts independent of PIF4 and PIF5. RVE1 regulates auxin levels by promoting its production during the day but not at night. It positively regulates the expression of auxin biosynthesis gene YUC8 to promote growth. Thus, YUC8 connects two signaling networks to coordinate plant growth with rhythmic changes in the environment (Rawat et al. 2009).

OsCCA1, a key clock component in rice, confers tolerance to drought, osmotic, and salinity stresses. There are 692 direct transcriptional target genes of OsCCA1 where, genes involved in ABA signaling are substantially enriched. OsCCA1 binds the promoters of OsPP108 and OsbZIP46 to activate their expression. Oscca1 null mutants display enhanced sensitivity to ABA signaling. Thus, abiotic stress adaptation by OsCCA1 is orchestrated by ABA signaling, linking the circadian clock with ABA signaling in rice (Wei et al. 2022). The OsRCAR10-OsABI5-OsPRR95 feedback loop regulates ABA signaling to fine-tune seed germination and seedling growth, indicating molecular link between ABA signaling and circadian clock in rice (Wang et al. 2022a).

Modifying circadian rhythms to develop climate-resilient productive crop cultivars

Novel variation in circadian clock systems

The presence of natural variation for circadian rhythms enables plant species to adapt to a wide range of latitudes with variable climates. The examples include variation in circadian period along a latitudinal gradient in annual population of common yellow monkeyflower (Mimulus guttatus) and domesticated soybean crop (Greenham et al. 2017); period length covaries with both geography and population substructure among Swedish Arabidopsis accessions (Rees et al. 2021); free running period (FRP)-dependent phase phenotype underlies photoperiod (short day) adaptation of Japanese duck weed (Lemna aequinoctialis) (Muranaka et al. 2022); and deceleration in circadian clock due to phase delay caused by allelic variation of the tomato homolog of the Arabidopsis EID1 expanded adaptation of domesticated tomatoes in farthest environment in Europe (long day photoperiod) far away from the center of origin (Müller et al. 2016).

Many crop wild relatives harbour genetic variation which enables them to flourish in harsh environments. Circadian rhythms in wild barley populations from ecogeographical locations of Southern Fertile Crescent in the Middle East, for example, revealed variation in circadian period length that correlated with temperature and aspects at the sites of plant origin, while the amplitudes of the rhythms correlated with soil composition. These data suggest that variation in environmental parameters exerts selection pressure on circadian rhythms and affects species adaptation (Dakhiya et al. 2017). A comparison of circadian clock rhythmicity and crop performance involving diverse barley lines (wild ancestors, landraces and breeding lines) under optimal and heat-stressed conditions revealed significant loss of thermal plasticity in circadian rhythms during domestication for output genes, while temperature compensation mechanisms was maintained in the core clock (Prusty et al. 2021). Domestication and artificial selection brought substantial changes in the developmental and circadian clock of plants possibly due to restructuring of plant metabolic timekee** (Siqueira et al. 2023). Wild relatives of domesticated crops are an important genetic resource for identifying and exploiting such variations in crop breeding. Rapeseed and wheat cultivars, widely grown, also show variations in circadian periods (Rees et al. 2019).

Daily rhythms of gene expression are critical to ensure that biological processes occur at the optimal time of the day (TOD) for traits of agricultural significance. A gene expression study in rice grown in the field environment and temporally monitored for an entire growing season revealed that most genes have a TOD-specific expression over the developmental time course. The thermocycle genes have stronger cue for TOD expression than the photocyclic genes. The core circadian clock genes, except for two grass paralogs of ELF3, display consistent TOD expression over the season, while ELF3 paralogs display distinct phases based on the interaction between thermo- and photo-cycles (Michael 2022).

The PRRs gene family in soybean exhibits rhythmic expression under LD and SD. The expression of most of the PRR genes is higher under LD in leaves. An investigating of the effects of natural variation in GmPRR alleles on soybean adaptation involving 207 germplasm accessions from China and USA. The majority of non-synonymous mutations in the coding region are associated with flowering time, while nonsense mutations—because of deletion of CCT domain—relate to early flowering (Wang et al. 2022b). Most of the Northeast China germplasm have haplotypes associated with early flowering, whereas those associated with late flowering are from lower latitudes. Thus, the PRR gene family in soybean provides a circadian clock-controlled flowering pathway and means to breed new germplasm adapted to specific daylength (Wang et al. 2022b).

Variation in circadian rhythm plasticity, denoting changes in period and amplitude under distinct temperatures, has been linked to both the maternal organellar genomes (plasmotype) and several nuclear loci in wild barley (Bdolach et al. 2019). Despite these findings, the molecular and biochemical basis of such cytonuclear interactions remain to be elucidated, as well as explored in other crops and models.

In short, assessing and exploiting functional allelic diversity in circadian rhythms and clock gene expression on a range of diversity panels of food crop germplasm may provide opportunity to tailor climate-resilient crops.

Photoperiod (or temperature) × circadian clock × early flowering gene interaction to enhance crops range adaptation

Climate change and variability can affect plant productivity. The increase in temperature can accelerate the reproductive phase in plant life cycle, thus reducing harvests (Parthasarathi et al. 2022). It has been noted that plant development affected by photoperiod uses gene networks that respond to changes in the light/dark cycle length (Osnato et al. 2022). Such circadian clocks, which are important for photoperiod-dependent flowering in high latitudes, are also influenced by temperature changes, thus triggering seasonal responses (Oravec and Greenham 2022). Adapting crops to changing climate in high latitudes, as evidenced in rice and wheat, must consider the interactions between photoperiod, temperature, and water availability.

Transcriptome to research has provided insights on increased expression due to high temperature of core clock genes such as CCA1, Gigantea-like protein (GI), PRR59, PRR73, PRR95, and HvLUX (an ortholog of the Arabidopsis circadian gene Lux Arrhythmo, also known as Phytoclock1) in barley (Ford et al. 2016). GI and PRR59 act rapidly when temperature increases, while both CCA1 and PRR73 respond when temperature decreases. The elf3 variant of barley lacks the GI and PRR responses due to temperature changes, suggesting that GI and PRR depend on a functional ELF3 (Ford et al. 2016). Thus, upregulation of core clock genes is pivotal in plants’ adaptation to temperature variations and the maintenance of circadian rhythms.

HvLUX1 (under purifying selection) seems to underly the early maturity 10 (eam10) mutant in barley (Campoli et al. 2013). The gene eam10 leads to circadian defects and interacts with the pseudo-response regulator Photoperiod-H1 (Ppd-H1) gene—a homolog of Arabidopsis AtPRR7—to speed flowering under both long and short photoperiods.

The emerging knowledge provides new insights for further genetic modification either through crossbreeding or genetic engineering. Modifying photoperiod through sexual hybridization, transgenics or gene editing will enlarge the range of adaptation in some daylength sensitive crops.

Severely compromised clock gene expression and clock outputs can favor crop adaptation

For breeders, circadian genes and networks have not been a major focus of artificial selection efforts. Despite this, many agronomic traits such as sensitivity to light/dark cycles (Creux and Harmer 2019; Okada et al. 2017), abiotic stress tolerance (Bonnot et al. 2021; Sharma et al. 2022), flowering and maturation time (Creux and Harmer 2019), plant architecture (Rubin et al. 2018), and yield (Steed et al. 2021) are controlled or affected by components of the circadian clock. Thanks to advances in comparative omics, it has been shown that multiple components of the circadian clock are altered in cultivated crops, when compared to their wild relatives (McClung 2021; Steed et al. 2021). This has occurred even though the domestication and breeding processes were performed without having any explicit focus or knowledge about circadian rhythms.

Recent research has been focused on understanding and manipulation of circadian clock components as tools for plant breeding and adaptation. For example, in tomato, a 3 bp deletion in the gene Empfindlicher imdunkelroten Licht protein 1 (EID1), which codes for a Phytochrome A-associated F-box protein, in addition to a near-complete deletion of Night Light-Inducible and Clock-Regulated 2 (LNK2; orthologue to AtLNK2) produces a lengthening of the clock period (Müller et al. 2016, 2018). These modifications enabled tomatoes from the equatorial region in South America to adapt from day-neutral circadian rhythms to the longer photoperiod of Europe and Mesoamerica (Müller et al. 2016, 2018). Another example of modifications in the circadian components, that allowed adaptation to high-latitude short-season regions, are mutations in the early maturity (EAM8) gene from barley (Faure et al. 2012) and in the earliness per se (Eps3) from wheat (Gawroński et al. 2014), which are homologs of the Arabidopsis thaliana EC genes, ELF3 and LUX, respectively (Fig. 2). These mutations in barley and wheat not only affect flowering time but also alter the circadian amplitude, period and phase (Faure et al. 2012; Gawroński et al. 2014). In a similar manner, allelic variations in O. sativa circadian heading-date genes (Hd genes not found in Arabidopsis), Grain Number, Plant and Heading Date 7 (Ghd7), Heading Date 1 (Hd1), Early Heading Date 1 (Ehd1) and Early Heading Date 4 (Ehd4) have contributed to adaptation of rice to higher latitudes (Cui et al. 2020; Gao et al. 2013, 2019). This could reveal that not only amplitude is an important factor in the resilience to changes in the environment, but also the plasticity of the rhythm itself.

Altered circadian rhythms and clock genes contribute to heterosis

Biomass and defense heterosis in Arabidopsis

Heterosis or hybrid vigor refers to the superior performance an F1 hybrid over its parents. Polyploidy (whole genome duplication) provides evolutionary advantages for plant growth, development, and adaptation. Some Arabidopsis allotetraploids and hybrids relative to their diploid parents (Arabidopsis thaliana and A. arenosa), can display vigour for morphological traits. Epigenetic modification of the circadian clock genes CCA1, LHY and their reciprocal regulators TOC1 and GI leads to changes in the expression of downstream genes and pathways. Epigenetic repression of CCA1 and LHY during the day induces TOC1, GI, and downstream genes harboring CCA1 binding site (CBS) 13. Allotetraploids and F1 hybrids produce more chlorophyll and starch than parents in the same environment. Single cca1 mutants, double cca1 lhy, and daily repression of cca1 in TOC1:cca1-RNAi transgenic plants, led to increased expression of downstream genes, chlorophyll, and starch content. Notably, constitutive expression of CCA1 or ectopically expressed TOC1:CCA1 had opposite effects. These studies suggest that hybrids and allopolyploids gain advantages via circadian-mediated physiological and metabolic pathways that lead to growth vigor and increased biomass (Ni et al. 2009). Parent-of-origin effects on biomass heterosis are associated with altered CCA1 expression amplitudes, which are associated with methylation levels of CHH (where H = A, T, or C) sites in the promoter region. The direction of rhythmic expression and hybrid vigor is reversed in reciprocal F1 hybrids involving mutants defective in the RNA-directed DNA methylation but not in maintenance methylation pathways. Such parent-of-origin effects on circadian regulation and heterosis is established during early embryogenesis and maintained throughout growth and development (Ng et al. 2014).

Plant immunity often causes growth-defense trade-offs. CCA1 confers heterosis for bacterial defense in hybrids without yield penalty, and even enhances the growth heterosis of hybrids under pathogen infection in Arabidopsis. Its genetic perturbation abrogated heterosis for both defense and growth in hybrids. The improved heterosis for defense and growth of hybrids at different time points (dawn, dusk) of the day is achieved when both resistance and growth are the most effective and have the least fitness cost. Plants promote defense heterosis prior to salicylic acid burst by rhythmically enhancing growth vigor during infections in a diurnal manner (Yang et al. 2021).

Yield heterosis in grain crops

A comparative assessment of transcript profiles of super-hybrid rice ‘LY2186’ and its parents at multiple time points for 2 day/night cycles identified 1552 rhythmic differentially expressed genes, RDGs (Li et al. 2021). The day-phased RDGs were associated with photosynthesis and the night-phased RDGs were involved in stress response, the major contributor to heterosis in ‘LY2186’. The circadian-related RDGs were core components in both phases (day and night) and primarily regulate downstream genes involved in photosynthesis, starch synthesis, plant hormone signal transduction, and other pathways. RDGs (72% of 282 RDGs) mapped onto yield-related QTL could be considered as candidate genes, which after functional validation, may be deployed in hybrid rice breeding (Li et al. 2021). An integrative profiling of metabolome and proteome in maize hybrids and their inbred parents revealed diurnal regulation of several metabolites and proteins. Also, key enzymes in photosynthetic and photorespiratory pathways and metabolites in C assimilation had nonadditive abundance and mild metabolic heterosis in hybrids. Amino acids display negative mid-parent heterosis (MPH), while sugars, alcohols, and nucleotide metabolites had positive MPH. The daily changes in metabolites and proteins, though small, may accelerate hybrid growth. Photosynthetic pathway metabolites have positive MPH, whereas photorespiratory pathway metabolites have negative MPH, which correspond to nonadditive protein abundance and key enzyme activities in the respective pathways (Li et al. 2020).

Greater levels of C fixation and starch accumulation are associated with altered temporal gene expression of diurnally upregulated ZmCCA1a in maize. CCA1a homologs, ZmCCA1a and ZmCCA1b, are diurnally upregulated in hybrids. ZmCCA1 expression complements the cca1 mutant phenotype in Arabidopsis. ZmCCA1b overexpression disrupts circadian rhythms and biomass heterosis due to reduced chlorophyll content and plant height. Reduced stem height results from reduced node elongation but not the total number of nodes. The height phenotype is less severe in the field than in greenhouse. Enhanced light and/or metabolic activities in the field compensates for altered circadian regulation, and activation of morning-phased genes in the hybrids promotes photosynthesis and growth vigor (Ko et al. 2016).

Circadian rhythms and clock genes mediating agronomic traits and abiotic stress adaptation

Transition from vegetative to reproductive phase, photoperiod-induced flowering, plant height, biomass and pod/seed traits, and storage roots in vegetatively propagated crops or tuberization are major agronomic traits influencing productivity of food crops. In addition to trait-responsive genes (i.e., core genes having identifiable direct impact on trait phenotype), several of these traits are also influenced by variation in circadian rhythms and clock genes.

Agronomic traits

Transition from vegetative to reproductive stage is critical to seed yield. Photoperiod pathways regulate flowering in rice, by synchronizing the exogenous environmental and the endogenous signaling cascades of photoreceptors, the circadian clock, and floral integrator genes. A diurnal rhythmic expression of OsLUX with the peak at dusk promoted flowering via the expression of genes associated with the circadian clock and the output integrators of photoperiodic flowering. OsLUX together with OsELF4a and OsELF3a or OsELF3b form two evening complexes (ECs), of which OsLUX-OsELF3a-OsELF4a predominantly promote photoperiodic flowering. OsELF4a alone also promotes flowering. Loss of OsELF4a, unlike OsLUX, marginally influences flowering under short day (SD), but markedly delays flowering under long day (LD) conditions (Cai et al. 2022).

Rice yields are predominantly determined by tillering. A regulatory loop involving the circadian clock, sugar, and strigolactone (SL) pathway regulates tiller bud and panicle development. OsCCA1, the circadian clock gene, positively regulates expression of SL pathway genes, OsTB1, D14 and IPA1 to repress tiller-bud outgrowth. OsCCA1 overexpression or downregulation, respectively, reduces or increases tiller numbers, while manipulating OsPRR1 expression causes opposite effects. OsCCA1 also regulates IPA1 expression to mediate panicle and grain development. OsTB1, D14, and IPA1 act downstream of OsCCA1. Sugars affect OsCCA1 negatively in roots and tiller buds but positively in the shoots to promote tiller-bud outgrowth. The circadian clock integrates sugar responses and the SL pathway to regulate tiller and panicle development (Wang et al. 2020a).

Optimizing crop duration to the available growing season is a major breeding target to match crop adaptation with the target environment. Variation in circadian-clock associated genes affects flowering in winter wheat cultivars in United Kingdom and European (Wittern et al. 2023). Early flowering 3 (ELF3) is a candidate gene for earliness per se (Eps) at D1 and B1 loci in wheat genome. A SNP within the coding region of TaELF3-B1 is the causal polymorphism at the Eps-B1 locus. Deletion of the Eps-D1 locus encompassing TaELF3-D1 is the basis of an allele that lies within an introgression region containing an inversion relative to the Chinese Spring D genome. Circadian rhythm is severely disrupted in emmer wheat (T. turgidum) cv. Kronos that carries loss-of-function alleles at TtELF3 locus, while loss of LUX in bread wheat, an Arabidopsis orthologue, also severely disrupts circadian rhythms. Thus, both ELF3 and LUX are part of the wheat circadian oscillator, where they integrate the wheat EC (Rees et al. 2022; Steed et al. 2021). Indeed, there exists sufficient allelic diversity within the three wheat ELF3 homoeologous to apply selection; i.e., delay or advance flowering, without adverse pleiotropic alterations to circadian rhythms in wheat (Wittern et al. 2023).

The ‘j’ allele in soybean delays flowering and enhances yield of long-juvenile soybean cultivars under SD conditions. ‘Huaxia-3’ (HX3) is a long-juvenile soybean cultivar with a loss-of-function allele j for the J gene. Transcriptome analysis of HX3 relative to the transgenic line overexpressing J gene unfolded 31 and 2311 differentially expressed genes, respectively, in HX3 under SD and LD conditions, with significantly enriched circadian rhythm pathway in HX3 under SD. GmELF3a and FT genes, GmFT2a and GmFT5a, were downregulated, while GmFT4 was upregulated under SD in HX3. FT homolog GmFT4, in contrast, was downregulated while GmFT1a was upregulated under LD in HX3, thereby suggesting that FT homologs may cause delay in flowering of long-juvenile soybean under SD (Sapey et al. 2022). A previous report using a soybean hairy root expression system to monitor endogenous circadian rhythms and the sensitivity of circadian clock to environmental stimuli revealed that a quadruple mutant deficient in GmLCLs orthologs resulted in extreme short-period circadian rhythm and late flowering in soybean. Thus, morning phased GmLCLs act constitutively to maintain rhythmicity, while their absence delays vegetative to reproductive phase transition in soybean (Wang et al. 2020b).

Plants with indeterminate inflorescences produce more flower structures than those with determinate type inflorescences. Decoupling floral primordia initiations from their maturation into grains would mean more resources available for the plants to invest in other important physiological functions. Flowering time genes in barley regulate initiation of floral primordia, while floral growth is specified by light signaling, chloroplast, and vascular development orchestrated by CCT MOTIF FAMILY 4 (HvCMF4), which is expressed in the inflorescence vasculature. Mutations in HvCMF4 increase primordia death and pollen failure by reducing rachis greening and limit energy supply to floral tissues. HvCMF4 senses light and connects with vascular-localized circadian clock to coordinate floral initiation and survival. Pyramiding beneficial alleles for both primordia number and survival may increase grain production in barley, caused by erratic stresses due to climate change and variability (Huang et al. 2023).

Abiotic stress adaptation

Several clock genes that impact circadian rhythms and abiotic stress adaptation are summarized herewith in diverse crops (Fig. 3). Melatonin (N-acetyl-5-methoxy-tryptamine) is a key hormone that functions as indole-3-acetic acid like hormones in mammals and plants, thereby regulating gene expression affects circadian rhythms, plant growth and development, and stress tolerance (Fan et al. 2018). Abiotic stress impacts the expression pattern of key melatonin biosynthetic genes. In rice, drought stress under light and dark conditions damages circadian clock genes, tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), and serotonin N-acetyltransferase (SNAT), but retains the functionality (circadian rhythm) of N-acetylserotonin O-methyltransferase (ASMT) gene in wild-type (WT) plants. Downregulation of ASMT expression in OsGI mutation suggests the involvement of the melatonin biosynthetic pathway in the OsGI-mediated circadian regulation. Thus, there exists a coordination between circadian rhythms and abiotic stress with respect to melatonin biosynthesis in rice (Ahn et al. 2021). However, more focused research on the role of melatonin on plant circadian clock may provide evidence-based mechanistic connection between melatonin and the circadian clock.

Fig. 3
figure 3

Circadian component effects under abiotic stress condition. This figure represents the effect of the mutation/silencing or overexpression of the circadian genes in the tolerance to different abiotic stresses. Solid lines represent direct regulatory effects while dashed lines represent indirect (or possibly direct but not experimentally determined) effects. Black lines ending with dashes represent the transcriptional repression of the circadian oscillator components. Green lines ending with arrows represent the transcriptional activation of the circadian oscillator components. Red lines ending with dashes represent transcriptional repression from or to the inputs and outputs in regard to the circadian oscillator. Blue lines ending with arrows represent transcriptional activation from or to the inputs and outputs regarding the circadian oscillator. The circadian repressor components in Arabidopsis are Circadian Clock Associated 1 (CCA1); Late Elongated Hypocotyl (LHY); Timing of CAB2 Expression 1 (TOC1); Pseudo-Response Regulator 9 (PRR9), PRR7 and PRR5; evening complex (EC). The circadian activation components in Arabidopsis are Reveille (RVEs); Night light-iducible and clock-regulated (LNKs); Light-regulated WD (LWDs); Teosinte Branched1-Cycloidea-PCE (TCPs). In Rice, OsCCA1 seems to transcriptionally activate protein phosphatase 2C (OsPP2C) and leucine zipper 46 (OsbZIP46), which have been shown to cope with the negative effects of ROS, drought, and salinity. OsPRR73 inhibit the High-affinity K + transporter 2;1 (OsHKT2;1) which is a negative factor in conferring tolerance to saline conditions. Similarly, the receptor for activated C kinase 1A (OsRACK1A) is circadian regulated and has been shown to have a negative effect increasing the sensitivity to salinity

Full size image

Drought accelerates leaf senescence which reduces canopy size, loss in photosynthesis, and reduced yields, while drought-induced delayed leaf senescence (i.e., stay green) contributes to crops fitness and productivity (Rivero et al. 2007). Variation in internal signals and environmental factors contribute to initiation and progression of leaf senescence (Guo et al. 2021; Zhang et al. 2021). ZmVQ52 is mainly expressed in maize leaves and is involved in the circadian pathway genes and photosynthetic pathways. WRKY family proteins (ZmWRKY20, ZmWRKY36, ZmWRKY50, ZmWRKY71) interact with ZmVQ52. ZmVQ52 overexpression accelerated pre-mature leaf senescence in Arabidopsis. Several leaf senescence genes in ZmVQ52 overexpression lines were upregulated by premature leaf senescence in Arabidopsis (Yu et al. 2019).

A large number of heat stress-responsive genes exhibit diurnal oscillation in Arabidopsis. Wild-type and mutants of the circadian clock genes (CCA1, LHY, PRR7, PRR9) exposed to heat (37 °C) and moderate cold (10 °C) during Zeitgeber (a rhythmically occurring natural phenomenon which acts as a cue in the regulation of the plant circadian rhythms) time ZT1 (early morning) and ZT6 (afternoon) revealed thousands of genes that were differentially expressed in response to temperature, the time of the day, or the clock mutation. In the morning, ~ 30% more genes were differentially expressed than in the afternoon. Heat stress significantly perturbed the transcriptome. Seventy percent of ~ 3000 DEGs showed time of the day (ZT1 or ZT6) occurrence of the transcriptional response. Changes in the magnitude of transcriptional response were detected in ~ 1400 DEGs shared between ZT1 and ZT6. About 2% of all DEGs had differential responses to temperature stress in the clock mutants. The clock genes CCA1 and LHY seem to have a more profound role than PRR7 and PRR9 in modulating heat stress responses during the day. Thus, time of the day plays a significant role in quantifying heat stress-responsive transcriptomes (Blair et al. 2019).

RACK1 is a WD40 type protein involved in multiple signaling pathways, conserved from prokaryotes to eukaryotes, and is involved in diverse biological functions and stress tolerance in plants (Adams et al. 2011; Zheng et al. 2017). OsRACK1A is a circadian rhythm gene involved in regulating salt tolerance in rice. OsRACK1A-suppressed transgenic rice compared to WT maintained low Na+ and high K+ concentrations, accumulate significantly more ABA and its transcripts and stress-inducible genes. Many stress-related genes, including AP2/ERF, were upregulated in OsRACK1A-suppressed transgenic rice (Zhang et al. 2018a). Another clock component, OsPRR73, positively regulates salt tolerance in rice by recruiting HDAC10 which transcriptionally repressed OsHKT2; 1, thus reducing cellular Na+ accumulation. osprr73 null mutants accumulated significantly higher levels of reactive oxygen species and sodium ions, which resulted in reduction in grain size and yield under salt stress (Wei et al. 2021).

Overall, circadian rhythmicity and clock genes that respond to abiotic stress, in turn interact with stress response genes to confer tolerance, providing broadened opportunities to develop more sustainable and productive cereal crops. In addition, several candidate clock genes with differential expression levels were identified as potential target for functional validation, for example, StGI, StPRR, and StEFM affecting tuberization in potato (Niu et al. 2022); COR expression in day and night impacting cold tolerance (Lu et al. 2021) and variable expression levels of morning (OsPRRs, OsLHY, OsZTL1) and evening (OsTOC1, OsGI, OsELF3) circadian clock genes in response to drought stress in rice (Li et al. 2017); HvELF3 expression impacting phase and shape of the clock and stress response (Habte et al. 2014) and variable expression of core clock genes (CCA1, GI, PRR59, PRR73, PRR95, and LUX) at higher temperatures (Ford et al. 2016) in barley; or circadian clock genes impacting significantly phase and amplitude in root and shoot under varying water regimes in the monocot model plant Brachypodium distachyon (Gombos et al. 2023).

Transgenic modification or genome editing to alter circadian rhythms to enhance abiotic stress adaptation and crops productivity

Modifying the circadian clock should be a breeding target to develop cultivars with higher buffering capacity to environmental stresses. Transgenic rice (japonica variety Taipei309) overexpressing CCA1 under the control of TOC1 promoter and containing RNAi construct A that follows circadian rhythm in progeny (T1, T2) plants has a detrimental effect on phenology and seed yield related traits over WT. CCA1 repression under the control of TOC1 promoter and containing RNAi constructs B and C that follows circadian rhythm in progeny plants significantly improve phenology and seed yield related traits over WT. Furthermore, RNAi construct C containing CCA1 derived from the 3’ terminal region of CCA1 generates plants with better phenology than construct B obtained from 5’ terminal region of CCA1. The progeny plants did not differ in chlorophyll content but had distinct rhythmicity, relative to CCA1 expression. Thus, CCA1 under the control of TOC1 promoter offers promise for development of crop cultivars better adapted to variable climates (Chaudhary et al. 2019).

CRISPR-Cas9 system has shown promise for plant breeding (Haque et al. 2018). Protein–protein interaction networks of differentially expressed proteins offer opportunities for better understanding of the organism response at the whole proteome level (Zou et al. 2018). A comparative proteomic analysis between CRISPR-Cas9 generated OsPYL9 loss-of-function mutants and WT at the whole proteome level revealed that CRISPR mutants had enhanced drought tolerance and improved grain yield under optimal and water-deficit conditions. Multiple proteins were differentially expressed (184 upregulated and 140 downregulated), with most of the upregulated DEPs related to circadian clock rhythm, drought response, and antioxidant activities in the mutants. DEPs were involved in response to abiotic stimulus and abscisic acid-activated signaling pathways. GIGANTEA, Adagio-like, and pseudo-response regulator proteins revealed higher interaction in protein–protein interaction (PPI) network. Both DEPs and higher PPI network in OsPYL9 mutant affect circadian rhythmicity, and a potential genetic resource and biomarker to breed for increased drought tolerance and improved yield in rice (Usman et al. 2020).

Overall, the results summarized in this section indicate the potential of genetic engineering for modifying circadian rhythmicity to enhance stress tolerance and productivity in food crops.

Managing circadian clock for breeding all seasons’ crops

The circadian clock that is based on interlocked negative loops of transcriptional activators and repressors is crucial for adjusting crop growth. Genes that maintain the plant circadian clock “ticking” (e.g. morning genes: CCA1, LHY; mid-day genes: PRR9, PRR7, PRR5; evening genes: TOC1, ELF3, ELF4, LUX) are supported by evidence in model plant Arabidopsis and other crops. Clock genes impacting biological rhythms and associated with plant growth, development, reproduction, and stress tolerance, have been identified in staple crops. Likewise, the role of phytohormones in mediating clock and stress-responsive gene expression is known. Maintaining the intricate interactions between circadian rhythms and clock genes‒phytohormones‒trait/stress-responsive genes may open new paths to tailor crops adapted to external environmental cues. Crop adaptation by changing flowering time is a good example of the potential of clock genes, where loss of some segments of circadian clock genes facilitated adaptation of tomatoes to distant growing habitats far from its center of origin (Müller et al. 2016, 2018).

The knowledge emerging on circadian clock molecular functioning can facilitate breeding of all seasons’ crops (Fig. 4). However, pursuit of such an approach poses a significant breeding challenge due to various factors; namely, (i) harnessing circadian oscillator genes that confer variation in biological rhythms in crop genepools, (ii) significant bottlenecks to establishing cost-effective high-throughput phenomics to assess variation in functioning of circadian oscillators in early breeding generations, (iii) possible negative trade-offs involving clock oscillator genes, phytohormones, abiotic stresses, and gene expression (i.e., trait- and stress-responsive genes), and (iv) identifying common genetic markers (e.g., SNPs, InDels and structural variation) linked with clock oscillators, phytohormones, and gene expression. Thus, assessing and exploiting functional allelic diversity in core oscillators as well as in trait-and stress-responsive genes can accelerate the development of climate-resilient productive and value-added germplasm for use in crop breeding.

Fig. 4
figure 4

The circadian oscillator in plants: integrating environmental and physiological cues to improve adaptation and resilience. The intricate circadian oscillator in plants continually integrates environmental and physiological cues, including signals from phytohormones, natural environments, and abiotic stress conditions. The oscillator responds to these inputs with coordinated output signals, allowing the plant to adjust its physiology and behaviour accordingly. Recent research has revealed that natural allelic variations or induced mutations in genes involved in the regulation of the circadian oscillator can significantly impact how plants perceive inputs or regulate outputs, ultimately leading to improved adaptation to new environments, enhanced tolerance to abiotic stress, and increased crop yields. This figure highlights the importance of understanding the genetic and molecular mechanisms underlying the circadian oscillator’s regulation and how they can be manipulated to improve plant performance and resilience

Full size image